Spatiotemporal mapping of RNA editing in the developing mouse brain using in situ sequencing reveals regional and cell-type-specific regulation

Abstract

Background: Adenosine-to-inosine (A-to-I) RNA editing is a process that contributes to the diversification of proteins that has been shown to be essential for neurotransmission and other neuronal functions. However, the spatiotemporal and diversification properties of RNA editing in the brain are largely unknown. Here, we applied in situ sequencing to distinguish between edited and unedited transcripts in distinct regions of the mouse brain at four developmental stages, and investigate the diversity of the RNA landscape. Results: We analyzed RNA editing at codon-altering sites using in situ sequencing at single-cell resolution, in combination with the detection of individual ADAR enzymes and specific cell type marker transcripts. This approach revealed cell-type-specific regulation of RNA editing of a set of transcripts, and developmental and regional variation in editing levels for many of the targeted sites. We found increasing editing diversity throughout development, which arises through regional-and cell type-specific regulation of ADAR enzymes and target transcripts. Conclusions: Our single-cell in situ sequencing method has proved useful to study the complex landscape of RNA editing and our results indicate that this complexity arises due to distinct mechanisms of regulating individual RNA editing sites, acting both regionally and in specific cell types.

Hybridization-based in situ sequencing (HybISS) for spatially resolved transcriptomics in human and mouse brain tissue

“…Larger gene panels (several fold increase) will be feasible with little to no adjustment to the method presented here. Additionally, as with previous ISS methods, HybISS and PLPs maintain the ability to detect single nucleotide variations due to their high specificity and sensitivity to be able to distinguish known point mutations ( 32 ) or editing sites ( 19 ) in tissues for example. SBL-based ISS has already shown its power in other non-neuronal tissue, in the form of cancer diagnostics ( 20 , 26 ) and tuberculosis granuloma ( 25 ) among others.…”

“…Multiplexed in situ hybridization offers the possibility to explore cellular diversity at subcellular resolution in an upscaled approach. As an example, our lab has developed in situ sequencing (ISS) to be used to detect RNA isoforms ( 19 ), transcriptomic distribution ( 20 ), and cell typing across tissue sections ( 21 ). The established ISS method based on barcoded padlock probes (PLPs) and amplification through rolling circle amplification (RCA) has shown robust detection of RNA for various applications, however, further improvements are needed to meet the upscaling demands to explore cellular diversity of scRNA-seq data across large tissue areas of various origins.…”

The spatial landscape of gene expression isoforms in tissue sections

“…While spatial profiling of specific isoforms within a tissue context has been demonstrated previously [2,3], these methods rely on a priori knowledge about transcript architecture. Here, we introduce Spatial Isoform Transcriptomics (SiT), an unbiased method based on spatial in situ capturing to detect and quantify spatial expression of splicing variants (Methods).…”

“…Such editing has been shown to be essential for neurotransmission and other neuronal functions [10]. While other studies have looked at editing events on bulk samples from mouse brain [11], or spatially resolved by ISS for a limited number of targeted editing sites [2], none has provided an exhaustive spatially-resolved RNA editing map. To this end, we performed additional sequencing for one of the CBS sections (CBS2) to achieve the necessary level of transcript information for robust calls of single nucleotide variants (SNVs) (Supplementary Table 1).…”

Hybridization-based In Situ Sequencing (HybISS): spatial transcriptomic detection in human and mouse brain tissue

“…Multiplexed in situ hybridization offers the possibility to explore cellular diversity at subcellular resolution in a upscaled approach. As an example, our lab has developed in situ sequencing (ISS) to be used to detect RNA isoforms 19 , transcriptomic distribution 20 , and cell typing across tissue sections 21 . The established ISS method based on barcoded padlock probes (PLPs) and amplification through rolling circle amplification (RCA) has shown robust detection of RNA for various applications, however further improvements are needed to meet the upscaling demands to explore cellular diversity of scRNAseq data across large tissue areas of various origins.…”